Objective:To analyze the effects of espO gene knockout on the biological characteristics of enterhemorrhagic Escherichia coli (EHEC). Methods:Two-step methods mediated by the suicide plasmid pCVD442-Δ espO and plasmid pTrc99a were used to construct the espO gene-deleted strain (Δ espO) and the complemented mutant (CΔ espO), respectively. HeLa cells were infected with different EHEC strains to analyze the biological functions and lethal effects of espO gene during infection. Results:PCR, electrophoresis and gene sequencing showed that the Δ espO and CΔ espO mutants were successfully constructed. Compared with the wild-type strain, neither the Δ espO nor CΔ espO mutant showed significant difference in growth rate, indicating that the espO gene had no influence on the growth and replication of EHEC. Furthermore, EspO could activate the tumor necrosis factor receptor (TNF)-induced NF-κB signaling pathway, while the effector protein NleB could inhibit the process. EspO could not inhibit the death of HeLa cells induced by TNF or TNF-related apoptosis-inducing ligand (TRAIL) after EHEC infection. Conclusions:In this study, we successfully constructed the espO gene-deleted and complemented mutants of EHEC and preliminarily analyzed the interaction between espO gene and host cells and the effects of espO gene on cell apoptosis during infection, which provided reference for further research on the in vitro biochemical activity and in vivo pathogenic roles of EspO.

Objective To investigate the application of venereal disease research laboratory(VDRL) test and toluidine red unheated serum test(TRUST) in syphilis laboratory diagnosis.Methods Serum,plasma and cerebrospinal fluid (CSF) in syphilis patients and healthy controls were measured by VDRL and TRUST.Results The VDRL detection results in serum and CSF sepcimens were generally higher than the TRUST detection results by 1-2 titers,while in plasma specimen,the VDRL detection results were generally lower than the TRUST detection results by 1-2 titers than TRUST when using plasma specimen.In addition,the VDRL detection in normal control serum and plasma specimens all appeared different degrees of false positive,but in the detection of normal control CSF,the results of TRUST and VDRL were consistent.Conclusion TRUST is more suitable for serum and plasma specimens,and CSF is suitable for the CSF specimen,but not suitable for serum and plasma detection.

Objective To investigate the expression of PD-1 on CD4 +and CD8 + T cells in patients with HBeAg-positive chronic hepatitis B(CHB)treated with telbivudine.Methods Fifty-six HBeAg-positive CHB patients admitted in the First Affiliated Hospital of Zhengzhou University during January 201 3 and June 201 4 were enrolled in this study.The expression of PD-1 on CD4 +and CD8 + T cells was detected with flow cytometry at baseline,24,48 and 72 wks after telbivudine treatment.The relationship of PD-1 expression with alanine aminotransferase (ALT)level,HBeAg seroconversion and HBV DNA loads was analyzed.t test and completely random variance analysis were used to analyze the data.Results The PD-1 expression on CD4 + and CD8 + T cells at baseline was higher in patients with low ALT levels compared to those with high ALT levels(t =1 2.20 and 9.69,both P <0.01 ),while higher levels of PD-1 expression was also observed in patients with high HBV DNA load (≥5 lgIU /mL)compared to those with low HBV DNA load (t =4.39 and 4.85,both P <0.01 ).PD-1 levels on CD4 + and CD8 + T cells presented a declining trend after telbivudine treatment(F =6.98 and 8.97,both P <0.01 ),PD-1 expression in patients with HBeAg seroconversion showed lower levels compared with baseline values (t =1 8.45 and 1 8.01 ,both P <0.01 ). Conclusion In HBeAg-positive CHB patients,the expression of PD-1 on CD4 + and CD8 + T lymphocytes shows a decreasing trend during the treatment with telbivudine,indicating that antiviral therapy may alleviate the immunosuppression in these patients.

BACKGROUND: Topoisomerase II alpha (TOP2A) has been reported to play a crucial role in the tumorigenesis of various cancer types. However, the biological role of TOP2A in gallbladder cancer (GBC) remains unknown. The current study aimed to explore the function and potential mechanism of TOP2A in GBC. METHODS: Based on Gene Expression Profiling Interactive Analysis data, we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival. Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues. In vitro, cell proliferation, migration, and invasion ability were examined by cell counting kit-8 and transwell assay, respectively. Epithelial-mesenchymal transition (EMT) related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway-related markers were measured by Western blotting. Xenograft model assay was performed to evaluate the effect of TOP2A in vivo. RESULTS: TOP2A was found up-regulated in GBC (tumor vs. normal, 12.62 vs. 0.34) and correlated with the late tumor node metastasis stage (P = 0.0032), present of lymph node metastasis (P = 0.0273), and poor prognosis in GBC patients (log-rank P = 0.028). In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation, migration, invasion, EMT process, and tumor growth in GBC. In addition, TOP2A down-regulation significantly decreased the protein levels of phosphor (p)-PI3K, p-Akt, and p-mTOR. CONCLUSION Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients. TOP2A promotes GBC cell proliferation, migration, invasion, EMT process, and tumor growth through activating PI3K/Akt/mTOR signaling pathway, and may serve as a novel prognostic biomarker and therapeutic target for GBC.

Objective The study was carried out to investigate the effects of different dietary Ca levels on bone metabolism, based on the osteoprotegerin ( OPG)-receptor activator of NF?κB ligand ( RANKL)?receptor activator of NF?κB ( RANK) system in growing WHBE rabbits. Methods Twenty one weaned male WHBE rabbits at the age of 42 days were divided into 3 groups (I, II and III) according to dietary Ca levels (0. 95%, 1. 10%, and 1. 30%, respectively) for a 42?d feeding trial. The above three diets had similar phosphor (P) (about 0. 64 g/kg), digestible energy (9. 50 MJ/kg), crude protein (about 19. 70%) and crude fibre (13. 57%) contents. When the feeding trial finished, the serum in?dices of bone metabolism (Ca, PTH and BALP) were detected, and the OPG?RANK?RANKL system in bone tissue was analyzed by real?time fluorescence quantitative PCR assay and immunohistochemistry, respectively. At last, the relationships between bone metabolism and dietary Ca were evaluated according to RANKL/OPG ratio. Results All the contents of serum Ca, PTH and BALP had no significant differences in the groups I, II and III (P>0. 05). Both the RANKL mR?NA and RANKL/OPG mRNA ratio were lowest in the group II and had significant differences with group I and III ( P<0. 05). Dietary Ca levels had significant effects on the protein expression of OPG?RANK?RANKL in bone tissues (P<0. 01). The positive index of OPG in the groups II and III was significantly higher than that in the group I(P<0. 01), while the positive index of RANK in the group II was lower than those of the group I and III(P<0. 01). The protein ex?pression positive index ratio of RANKL to OPG was also lowest in the group II, showing a significant difference with group I(P<0. 01). Furthermore, both the gene transcription ratio of RANKL to OPG and the protein expression positive index ratio of RANKL to OPG had significant correlations (with quadratic curve) to the dietary Ca levels (R2 =0. 4068, 0. 8433;P<0. 05,P<0. 001). Conclusions In summary, the bone metabolism of WHBE rabbits during growing periods has sig?nificant correlation with dietary Ca levels. An optimal bone metabolism status can be obtain at 1. 10% dietary Ca level as demonstrated in this study.

Objective To evaluate the efficacy of quadrates lumborum block for unilateral inguinal hernia repair in elderly patients. Methods Fifty-eight elderly patients with unilateral inguinal hernia of both sexes, aged 65-80 yr, with body mass index of 18-25 kg∕m2 , of American Society of Anesthesiolo-gists physical status Ⅱ or Ⅲ, scheduled for elective unilateral tension-free repair, were divided into 2 groups ( n=29 each) using a random number table method: iliohypogastric-ilioinguinal nerve block group (group T) and quadrates lumborum block group (group Q). Iliohypogastric-ilioinguinal nerve block with arteria circumflexa ilium profunda as a marker was carried out with 0. 33％ ropivacaine 20 ml under ultra-sound guidance in group T. The anterior approach to quadratus lumborum block was performed with 0. 33％ropivacaine 20 ml under ultrasound guidance in group Q. Operation was started after the height of sensory block was assessed by pin-prick test at 30 min after block. When the blocking effect did not meet the opera-tion requirements, an increment of 1％ lidocaine 2. 5 ml was given every time in the surgical field until op-eration requirements were met. Dexmedetomidine was intravenously infused at a rate of 0. 03-0. 07μg·kg-1 ·min-1 during surgery until the end of surgery to maintain Narcotrend index between 80 and 90. When postoperative visual analogue scale score >3, parecoxib sodium 40 mg was intravenously injected, and if marked pain relief was not found 10 min later, tramadol hydrochloride 50-100 mg was intravenously injected. The upper spread of sensory block and intraoperative requirement for additional local anesthetics were recorded at 30 min after nerve block. The requirement for parecoxib and tramadol was recorded within 48 h after operation. The development of inadvertent intravascular injection of local anesthetics, local anes-thetic intoxication and postoperative nausea and vomiting, nerve block of lower extremity and uroschesis was recorded. Results Skin pain disappeared at the plane of T11-L1 in group T and at the plane of T9-L1 in group Q. Compared with group T, the intraoperative requirement for and consumption of local anesthetics, postoperative requirement for parecoxib and tramadol, and postoperative incidence of nausea and vomiting were significantly decreased in group Q ( P<0. 05) . Conclusion Quadrates lumborum block provides bet-ter efficacy for unilateral inguinal hernia repair than iliohypogastric-ilioinguinal nerve block in elderly pa-tients.

Objective:To establish a new molecular typing method for Treponema pallidum (TP) based on TP0136 protein sequence heterogeneity. Methods:The amino acid sequences of TP0136 open reading frame (ORF) of 9 strains of Treponema pallidum ssp. Pallidum (TPA) , 3 strains of Treponema pallidum ssp. Pertenue (TPE) , 1 unclassified simian strain of Treponema Fribourg-Blanc (FB) and 1 strain of Treponema pallidum ssp. Endemicum (TEN) were searched from Genbank, and multiple sequence comparisons were performed to obtain the molecular typing results of TP0136 protein. The TP0136 protein-based molecular typing method was used to classify 23 TPA clinical isolates, which were collected from Dermatology Hospital of Southern Medical University from January 2015 to December 2018, and the typing results were compared with those by the traditional typing method based on the tp0548/Arp/Tpr genes. Results:TP0136 protein was highly heterogeneous in different TP strains. According to the amino acid sequence of TP0136, TPE, FB and TEN strains were divided into 4 subtypes of Ⅰ- Ⅳ, TPA strains were divided into 6 subtypes of Ⅴ-Ⅹ, and TPA clinical strains were classified into 4 subtypes of Ⅶ, Ⅸ, Ⅹ, Ⅺ. Through the traditional typing method described above, 23 TPA clinical strains could be divided into 5 types (13D/d, 14D/f, 14D/g, 15D/f, 16A/e) . By using the TP0136 protein-based typing method combined with traditional typing method, the above clinical strains could be further subdivided into 10 types, and the 14D/f type could be further divided into 3 subtypes by using the TP0136 protein-based typing method.Conclusion:The TP0136 protein-based molecular typing method can be used to distinguish TP species, which is helpful for further improvement of traditional TPA molecular typing.

Objective To isolate and culture a clinical strain (GDI) of Treponema pallidum (Tp) in Guangdong province,and to investigate the difference in nonsynonymous single nucleotide polymorphisms (nsSNPs) between the GD1 strain and Tp Nichols strain.Methods The GD1 strain was isolated from the hard chancre in a patient with primary syphilis in Guangdong province,and continuously subcultured in the testes of New Zealand white rabbit.The serial subcultivation of GD1 was multi-verified by dark-field microscopy,polymerase chain reaction (PCR) for TP0548 gene,DNA sequencing and genotyping.Meglumine diatrizoate density gradient centrifugation was performed to isolate rabbit tissues and concentrate GD1,and DNA sequencing was used to verify the nsSNPs in the TP0443 and TP0584 genes.Results The GD1 strain was successfully isolated from the lesions of the patient with syphilis,and classified as a subtype f of TP0548.Compared with the American Tp (Nichols strain),there were nsSNP mutations in the GD1 strain.One mutation was located in the TP0443 gene,leading to the the substitution of threonine by alanine at amino acid position 120,and another one was located in the TP0584 gene,which caused a change from alanine to threonine at amino acid position 314.Conclusion The GD1 strain was successfully isolated firstly from the lesions in a patient with syphilis in Guangdong province,and nsSNP mutations were confirmed in the GD 1 strain on the etiology.

Objective To investigate the expression of TP 0155 mRNA in rabbits with early infection of Treponema pallidum ( T.pallidum ). Methods Three New Zealand white rabbits were subcutaneously injected with T.pallidum Nichols Seattle strains.Each rabbit was inoculated at ten sites with 106 T.pallidum/site.Skin lesions at the primary stage of syphilis were observed at different time points. Biopsy from one of the lesions was obtained from each rabbit every three days for detection of TP 0155 mRNA and house keeping gene TP 0574 mRNA.TP0155 plasmid standard was constructed by molecular cloning technique , and the quantitative PCR was used to continuously detect the expression of TP 0155 mRNA and TP0574 mRNA from lesion at different time points.Kruskal Wallis test and Bonferroni method were used to analyze the data.Results On d6, red papules appeared on the dorsal skins of rabbits ,there were ulcers in the center of the lesions on d19,presenting typical appearance of syphilis chancre.On d24 the scab of ulcer became smaller; on d25 the rabbits showed disseminated secondary syphilis , which became smaller and disappeared on d30.The copy numbers of TP0155 plasmid standards were 7.48 ×109 copies/μL.There were significant differences in expression of both TP 0155 mRNA and TP0574 mRNA at different time points (χ2 =32.756 and 52.344,both P<0.01).The expression levels of TP0155 mRNA and TP0574 mRNA increased in the early stage, and both reached the peak at d15 (both P<0.05), and then rapidly declined. There were significant differences in normalized TP 0155 mRNA ( TP0155 ×1000/TP0574 mRNA ) at different time points(χ2 =19.758,P<0.05),which reached the peak on d24 and d30,respectively (all P<0.05).Conclusion The level of TP0155 mRNA increases with the disappearance of chancre and secondary syphilis lesions , suggesting that TP0155 might be involved in immune escape of T.pallidum.

Objective To evaluate the efficacy and safety of epalrestat, an aldose reductase inhibitor, and epalrestat plus methylcobalamine on diabetic peripheral neuropathy, as compared with methylcobalamine. Methods A total of 444 subjects with diabetic neuropathy were enrolled in the study, and divided into methylcobalamine group ( n= 145 ) , epalrestat group ( n = 143 ) , and methylcobalamine combined with epalrestat group ( n = 156 ) . Therapeutic efficacay was assessed in terms of clinical symptoms and physical examinations by using Michigan Neuropathy Screening Instrument ( MNSI ) , and electrophysiological assessments. Results After 4 to 12-weeks′treatment, symptoms and signs of neuropathy ( using MNSI ) are significantly improved in the three groups ( P<0. 01). The mean changes of MNSI ( questionnaire) score from baseline were higher in epalrestat group and methylcobalamine combined with epalrestat group as compared with that of methylcobalamine group(P<0. 05), but no difference was detected in the change of MNSI ( physical examination ) score from baseline among three groups. After treatment for 12 weeks, motor nerve conduction velocity ( MNCV ) was significantly improved in epalrestat group and methylcobalamine combined with epalrestat group(P<0. 05), but no difference was detected in MNCV at 12 week among three groups(P>0. 05). Conclusion Epalrestat is effective and safe in the treatment of diabetic neuropathy. Furthermore, epalrestat is more efficacious in ameliorating symptoms and MNCV of neuropathy than methylcobalamine. However, while no improved efficacy is shown with the combined treatment.